Multi-target directed (targeted) approach for transposon tagging
--Guri Johal and D. Gruis

Transposon tagging is the most efficient method for cloning genes in maize that are known only by their mutant phenotypes. Dozens of genes have been cloned by this method over the last 10-12 years. However, the success rate of this method has been depressingly low. In addition, the method requires several years for completion, and is very labor- and resource-intensive. As a consequence, transposon tagging has been considered more of an adventure than a method to rely upon for routine isolation of genes.

One reason for these drawbacks is that often a single locus is used as a target in a tagging project. Typically, a tester stock, homozygous recessive for a gene of interest, is either developed or acquired from the maize Co-op or a fellow scientist and is crossed mostly as a pollen parent to a stock carrying the transposons. Since only a single locus (target) is present in the homozygous tester, mutations involving only that locus are uncovered in the F1, unless the mutational event generates an allele that shows dominance over the wild-type allele. Such dominant mutations are however very rare, at least 100 times less frequent, compared to recessive mutations.

In order to increase the utility of transposon tagging we asked if it would be possible to tag more than one gene in one mutagenesis experiment. We found that such a strategy is possible. In a stock showing transposable element activity, transposons can and probably do insert into genes throughout the genome, and the gametes produced by this stock may contain the entire set of tagged genes. However, a mutation will be generated, or more appropriately recovered, in a gene for which the gametes coming from the tester parent are also defective. If these gametes are mutant for more than one locus, plants showing mutant phenotypes for all of these loci can be obtained in a single experiment. We proved this contention in a tagging experiment where Mutator active stocks were crossed with the Mangelsdorf's tester. This multiple gene tester is homozygous recessive at ten different loci, one on each of the ten chromosomes. An F1 progeny of 60,000 was planted last summer in Columbia and mutant plants showing phenotypes for each of the ten recessive loci were recovered. All of these mutants are available upon request.

The frequency of mutation, however, was vastly different at different loci. Compared to more than 15 mutants for both brown midrib2 (bm2) and japonica1 (j1), only one golden1 and two liguleless1 (lg1) mutants were found in this progeny. Most of our Mutator stocks were acquired from Steve Briggs (Pioneer Hi-Bred Int'l Inc.) and Pat Schnable (Iowa State University), and the progeny of each stock was kept separate from each other. Judging from mutations at the bm2, j1 and a1 loci, it was realized that mutation frequency of these loci was stock-dependent. Some stocks were more efficient, 10-20-fold compared to others, in generating mutations at the bm2 locus, and similar stock-dependent mutation efficiency was also evident for the a1 and j1 loci. Presently, it is unclear why different Mutator stocks differ in their ability to cause mutations at different loci, but it is possibly the result of uneven distribution of Mu elements on individual chromosomes in different stocks. A clustering of Mu elements has been reported (Ingels et al., J. Heredity 83:114, 1992). Although not shown for Mutator yet, transposition to linked sites is one mechanism that can cause clustering of Mu elements and also the bias among stocks for generating mutations at different loci in the genome.

In summary, the genetic scheme described above can allow the tagging and cloning of multiple genes in a single project and can change transposon tagging from an adventure into a highly rewarding venture. Although we used a tester that was homozygous recessive for multiple loci, a tester heterozygous for many genes will also work. The only drawback with a heterozygous tester is that half of the tagged mutant alleles will not be recovered. However, if we can identify chromosome specific Mutator stocks that can produce mutations at a frequency of 1x10-4, losing half of the tagged gametes will have an insignificant effect on the success of the tagging project. 

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